Clinical Pathology Laboratory Report by Kula Jilo 2016

Clinical pathology laboratory report by KULA JILO 2016

Jimma University College of agriculture and veterinary medicine

School of veterinary medicine

Clinical Pathology Laboratory Report

Submitted by: Kula Jilo

ID.NUMBER=05537/04

Submission date: 13-01-2016

Submitted To: mr.Eshetu (lab.technician)

January, 2016

Jimma, Ethiopia


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Laboratory Manual and Review on Clinical Pathology

Abstract

Clinical pathology is a subspecialty of pathology that deals with the use of laboratory methods

(clinical chemistry, microbiology, hematology and emerging subspecialties such as molecular

diagnostics) for the diagnosis and treatment of disease. Hematology studies the blood and

blood-forming tissues to evaluate presence of disease and assist in therapeutic interventions as

clinically indicated. Clinical chemistry (also known as chemical pathology and clinical

biochemistry) is the area of clinical pathology that is generally concerned with analysis of bodily

fluids. Some of the objectives of this manual are to identify the most important hematological

and functional pathological tests of vet importance, to diagnose different animal diseases by

confirming the pathological causes that constraint livestock production and to have knowledge

more about clinical pathology. Part one discusses about hematology which includes equipment’s

and reagents, blood collection sites and procedures, preparation method for working solution,

staining methods (staining procedures), hemoglobin determination, hematocrit determination

(PCV), total RBC count, total WBC count, differential leukocyte count, determination of ESR,

coagulation time determination, bleeding time, calculating red blood cell indices and blood

group and Rh factor determination. Part two deals with function tests which includes

determination of Aspartate Amino Trasferase (AST) and Glutamic OxalacetateTransminase

(GOT), determination of Alkaline Phosphtase (ALP), determination of creatinine, total protein

determination, urea determination, total and direct bilirubin determination, enzymatic kinetic

colorimeter test, liver function test, kidney function test, rumen function test and pancreatic

function test. In general, the outline of this laboratory manual deals with the basic

hematological procedures and clinical chemistry analysis.

Objectives of Clinical Pathology Practical Course

Objectives of this course are enabling students to:

a) Identify the most important hematological and functional pathological tests of vet

importance

b) Diagnose different animal diseases by confirming the pathological causes that

constraint livestock production in general

c) Have knowledge more about clinical pathology

d) Write lab report pertaining the tests carried out


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Equipment’s and reagents required for hematological blood examination.

Hematocrit centrifuge

Compound microscope

Sahlis instrument

Capillary tube

Hematocrit reader

Distilled water

Haemocytometer pipette

Haemocytometer counting

chamber

WBC diluting fluid

RBC diluting fluid

Slides cover slip

special cover slip

ESR stand

ESR tube

Filter paper

blood lancets

lead pencils

marker

Giemsa stain

Wright’s stain

Vacutainer tubes(different

type)

vacutainer needles

syringes

needle holder

Gloves

Blood samples

Staining racks and others

Lab.2. smear preparation

Procedures:

Blood was collected from jugular vein of animal (cow) with EDTA Vacutainertube.Then

collected blood is transported to the laboratory and wet smear, thin smear and thick smear

were done respectively.

I. Wet blood smear preparation

1. A drop of blood was placed at the center of a clean slide

2. Covered with a clean, dry cover slip

3. Then examined under the microscope (40 × objective).

Result: blood cells structures observed and parasites checked

II. thin blood smear preparation

1. A drop of blood made on one end of glass slide

2. A drop of blood was evenly spreaded across a clean grease free slide using a

smooth edged spreader holding at angle of 45 degree

3. Then smear was dried by waving rapidly in the air

4. Then examined under the microscope (40 × objective).


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Result: different blood cells observed

III. Thick bloods smear preparation

1. A large drop of blood was put at the center of a clean dry slide

2. The drop was spreaded with corner of another slide to cover an area

of ½ an inch square

3. The smear is thoroughly dried in a horizontal position so that the

blood could not ooze to one edge to the film and protected from

dust, insects and direct sunlight.

Result: RBC observed

Lab. 3 Staining Dyes

Dry powder as bulk and as pre weighted vials, Capsule Readymade

solution and most convenient, but often deteriorated from long storage.

3.1. Preparation of stain

Conventional method

1. 0.5gm of dry Wright’s stain powder was placed in a mortar 2. 300ml of absolute methyl alcohol was added 3. The mixture and pour was grinded in dark bottle 4. The bottleshacked each day for about two weeks, and then filtered. 5. Aging of the stain took 3 weeks for use

Accelerated method

1. 0.3gm of dry Wright’s stain powder was placed in a mortar and over

layed with 3ml of glycerol and grinded thoroughly

2. Rinsed with 100ml of absolute methyl alcohol and placed in dark

container

3. Mixed with magnetic stirrer for about 1 week without heat

4. then filtered and used


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3.2. Giemsa Stain

General consideration

Giemsa stain has various azure compounds with eosin and methylene blue. It is an

excellent stain for blood parasites and for inclusion bodies it stains red granules

well but neutrophilic granules and erythrocytes are poorly stained. Commercial

stock solution are recommended for purchase and are stable indefinitely

Preparation method for working solution:

1. one part of giemsa stock solution and nine part distilled water was

taken

2. And mixed before to two hours and used.

3.3. Leishmen’s stain

Preparation

1. 0.15gm of Leishmen’s stain powder was grinded with small amounts

of absolute methyl alcohol until an even suspension is obtained.

2. Then a total of 100ml methanol is added to produce complete

solution

3. Then Poured in to a dark bottle

4. and age for a few weeks prior to used

3.4. Wright’s-Giemsa stain (modified Wright’s stain)

Preparation of stain

1. 500mg of dry Wright’s stain powder and 50mg of Giemsa stain

powder were ground in a mortar with 100ml of absolute methyl

alcohol (acetone free)

2. Allowed to stand for 24-48 hrs. before used

3. Kept with well stoppered to prevent evaporation of alcohol or

absorption of water vapor

Commercial Wright’s – Giemsa stain are available

• Wright – Leishmen stain

• May – Grunwald –Giemsa stain

• Methylene blue stain

• Field’s stain


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• Prussian blue stain and others

3.5. Buffered distilled water

Preparation:

5.47g monobasic potassium phosphate and 3.8g of dibasic sodium

phosphate were mixed with 1000ml of distilled water

Methods of staining (staining procedure)

a) Wright’s stain

1. The dried blood smear is covered completely with Wright’s stain and

allowed to stand for 1 to 3 minute

2. An equal amount of buffered distilled water or neutral water (pH 6.6

to 6.8 for most animals blood) was added

3. Blown gently and allowed the mixture to stand for 3 to 5 minutes.

4. The metallic scum with a stream of water from a wash bottle was

floated from the top.

5. Distilled water was used.

6. The stain wiped from the back of the slide with cleaning tissue

7. Standing the slide on end and waved gently in the air to dry

8. then preparation examined by placing a drop of oil on a slide

Advantage: It conserves stain, Methyl alcohol, and time

Disadvantage: More precipitate is likely to be present

Cell Cytoplasm Chromatin

Erythrocytes Yellow to pinkish Purple

Lymphocytes Blue Purplish red

Monocytes Light blue Purple

Eosinophils Yellow to brownish red rods Light purple

Basophils Dark purple granules

Hetrophils/Neutrophils Yellow to brownish red rods Light purple

Thrombocytes Grey-blue Purple

Table 2: Staining characteristics.


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b) Giemsa stain method

1. Place dried blood smear in a coplin jar containing absolute methyl

alcohol for about 3 minute to fix the smear

2. Drain off the alcohol and allow the slide to dry

3. Transfer the slide to a second coplin jar containing fresh stain and

allow staining for 16 to 60 minute. 60 minute staining period is used

when blood parasite is suspected. 12 to 18 hrs for inclusion bodies

4. Wash the stain with running/tap water, dry and examine under oil

immersion microscope

c) Leishman’s stain method

1. the air dried blood film flooded with Leishman’s stain and stayed for

1 to 2 minute to fix

2. the stain diluted on the smear with double the volume of buffered

distilled water and stain for 5 to 15 minute

3. Blew on the mixture gently

4. Washed with distilled water until the film has a pinkish tinge

5. the back of the stain/the slide Wiped and allowed to dry in upright

position

Lab.4 and 5 Hemoglobin Determinations

Objective:

To determine the amount of hemoglobin present in 100 ml of blood of

a given sample.

Significance

a. It serves as an index of blood condition of the animal.

b. If the hemoglobin [Hb] content falls below the normal levels, it indicates

anemia, or pregnancy (physiological).

c. If it increases to the normal value, it indicates polycythemia, decrease in

O2 supply, heart disease, emphysema etc.

Method: Acid hematin method

Requirements: Sahlis instrument, blood sample

Procedure

1. 0.1N HCl (1%) was taken into central graduated tube up to mark 2.


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2. The blood sucked exactly up to mark 20 (20 μl) with the help of sahlis

pipette.

3. The blood transferred from pipette to central graduated tube of the

hemometer.

4. Then mixed well with the help of stirrer or rod and allowed it to react

for two minute.

5. The distilled water was added drop by drop until the color matches

with the standard comparator tube and mixed well.

6. After the color matched it taken out and recorded the values on the

side as gm/100ml and or in percentage.

7. Repeated 5 to 6 times and the average value was taken

Normal value:

Bovine 8 - 15gm/dl (11) Equine 11 -19gm/dl (14)

Ovine 9 – 15gm/dl (11.5) Feline 8 – 15gm/dl (12)

Caprine: 8-14gm/dl (11) Canine 12 – 18gm/dl (15)

Hematocrit Determination (PCV)

Aim: To determine the hematocrit value for a given blood sample.

Principle: Blood compartment is separated into three parts using capillary tube in

a hematocrit centrifuge.

Significance

• Packed Cell Volume (PCV) = erythrocyte mass; anemia when PCV falls dawn.

• Buffy coat; white to gray layer above PCV. It will give number of WBC (0.5mm

to1.5mm).Leukopenia or leukocytosis.

• Plasma content: usually about 55%, Yellowish in color. Degree of yellowness

indicates icterus (jaundice).

Requirements: Hematocrit tube, hematocrite centrifuge, hematocrit reader and

sealer.

Procedure

• The blood was filled in to a micro hematocrit tube (3/4th) and it sealed with

sealer.

• The filled hematocrit tube in a hematocrite centrifuged at 2000 rpm for 4-5

minutes.

• Then the value (the tube) with hematocrit reader was read and recorded.

Normal value

• Bovine 37 - 55 % (45%)

• Ovine 24 - 50 % (38%)

• Caprine 24 - 50 % (40%)

• Equine 32 - 55 % (42%)

• Swine 32 - 50 % (42%)


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Lab.6 and 7

Total Count of RBC

Objective: To enumerate the total count of RBC/cumm of a given blood sample.

Method: Hemocytometry method

Significance

• It performs some functions such as transportation of O2 and CO2

• A decrease in RBC accounts for less hemoglobin i.e., anemia

• An increase in RBC is referred as Polycythemia

Requirements: Hemocytometer, cover slip, microscope, RBC diluting fluid,

Haeyem’s solution or Physiological saline 0.85% Nacl.

Procedure

1. the blood is taken in to RBC pipette up to 0.5 marks

2. The RBC diluting fluid was drawn up to mark 101.

3. The pipette was rotated between thumb and other fingers with finger eight

(8) movements. This gives a dilution of 1:200.

4. the counting chamber of hemocytometer and cover slip was cleaned

5. cover slip placed in position over the counting chamber by gentle pressure

6. A drop of blood on to the counting chamber was expeled by holding the

pipette at an angle of 45º.

7. hemocytometer allowed for 2-3 min to settle down the RBC in counting

chambe

8. Counted less than 40 × microscope objective

9. Cells touched the left and top side lines were not counted.

10. Cells touching the bottom right side lines were not counted.

11. Cell counted first left to right direction, then to vise verse.

*******THE END******

Clinical Pathology Laboratory Report by Kula Jilo 2016

Education

Transcript of Clinical Pathology Laboratory Report by Kula Jilo 2016